Methods for treating b cell lymphoma using CD80-specific antibodies

ABSTRACT

The present invention relates to methods for treating B cell lymphoma using CD80-specific antibodies.

RELATED APPLICATIONS

This is a divisional application of application Ser. No. 09/383,916,flied Aug. 26, 1999, now U.S. Pat. No. 6,709,654 B1, which is adivisional application of application Ser. No. 08/487,550, filed Jun. 7,1995, which issued as U.S. Pat. No. 6,113,898.

FIELD OF THE INVENTION

The present invention relates to the manufacture and identification ofnovel monoclonal antibodies to human B7, i.e., human B7.1 and human B7.2and primatized forms thereof. More specifically, the present inventionrelates to the production and identification of macaque antibodies tohuman B7, i.e., human B7.1 and human B7.2 produced by screening of phagedisplay libraries and monkey heterohybridomas using B lymphocytesobtained from B7 immunized monkeys.

The invention further relates to specific primatized antibodies whichbind to human B7, i.e., human B7.1 and B7.2 as well as theircorresponding amino acid and nucleic acid sequences.

Also, the present invention relates to pharmaceutical compositionscontaining monkey monoclonal or primatized antibodies specific to humanB7.1 and/or human B7.2 and their use as immunosuppressants by modulatingthe B7:CD28 pathway, e.g., for the treatment of autoimmune disorders,and the prevention of organ rejection.

BACKGROUND OF THE INVENTION

The clinical interface between immunology, hematology, and oncology haslong been appreciated. Many conditions treated by the hematologist oroncologist have either an autoimmune or immunodeficient component totheir pathophysiology that has led to the widespread adoption ofimmunosuppressive medications by hematologists, whereas oncologists havesought immunologic adjuvants that might enhance endogenous immunity totumors. To date, these interventions have generally consisted ofnonspecific modes of immunosuppression and immune stimulation. Inaddition to the limited efficacy of these interventions, toxicitiessecondary to their nonspecificity have also limited their overallsuccess. Therefore, alternative strategies have been sought.

Elucidation of the functional role of a rapidly increasing number ofcell surface molecules has contributed greatly to the integration ofimmunology with clinical hematology and oncology. Nearly 200 cellsurface antigens have been identified on cells of the immune andhematopoietic systems (Schlossman S F. Boumsell L. Gilks J M, Harlan T.Kishimoto, C. Morimoto C, Ritz J. Shaw S, Silverstein R L, Springer T A,Tedder T F, Todd RF:CD antigens (1993), Blood 83:879, 1994). Theseantigens represent both lineage-restricted and more widely distributedmolecules involved in a variety of processes, including cellularrecognition, adhesion, induction and maintenance of proliferation,cytokine secretion, effector function, and even cell death. Recognitionof the functional attributes of these molecules has fostered novelattempts to manipulate the immune response. Although molecules involvedin cellular adhesion and antigen-specific recognition have previouslybeen evaluated as targets of therapeutic immunologic intervention,recent attention has focused on a subgroup of cell surface moleculestermed co-stimulatory molecules (Bretscher P: “The two-signal model oflymphocyte activation twenty-one years later.” Immunol. Today 13:73,(1992); Jenkins M K, Johnson J G: “Molecules involved in T-cellco-stimulation.” Curr Opin Immunol 5:351, 1993; Geppert T, Davis L. GurH. Wacholtz M. Lipsky P: “Accessory cell signals involved in T-cellactivation.” Immunol Rev 117:5, (1990); Weaver C T, Unanue E R: “Theco-stimulatory function of antigen-presenting cells.” Immunol Today11:49, (1990); Stennam R M, Young J W: “Signals arising fromantigen-presenting cells.” Curr Opin Immunol 3:361, (1991)).Co-stimulatory molecules do not initiate but rather enable thegeneration and amplification of antigen-specific T-cell responses andeffector function (Bretscher P: “The two-signal model of lymphocyteactivation twenty-one years later.” Immunol. Today 13:73, (1992);Jenkins M K, Johnson J G: “Molecules involved in T-cell co-stimulation.”Curr Opin Immunol 5:351, (1993); Geppert T, Davis L. Gur H. Wacholtz M.Lipsky P: “Accessory cell signals involved in T-cell activation.”Immunol Rev 117:5, (1990); Weaver C T, Unanue E R: “The co-stimulatoryfunction of antigen-presenting cells.” Immunol Today 11:49, (1990);Stennam R M, Young J W: “Signals arising from antigen-presenting cells.”Curr Opin Immunol 3:361, (1991); June C H, Bluestone J A, Linsley P S,Thompson C D: “Role of the CD28 receptor in T-cell activation.” ImmunolToday 15:321, (1994).

Recently, one specific co-stimulatory pathway termed B7:CD28 has beenstudied by different research groups because of its significant role inB and T cell activation (June C H, Bluestone J A, Linsley P S, ThompsonC D: “Role of the CD28 receptor in T-cell activation.” Immunol Today15:321, (1994); June C H, Ledbetter J A: “The role of the CD28 receptorduring T-cell responses to antigen.” Annu Rev Immunol 11:191, (1993);Schwartz R H: “Co-stimulation of T lymphocytes: The role of CD28,CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy.” Cell71:1065, (1992)). Since this ligand:receptor pathway was discovered fouryears ago, a large body of evidence has accumulated suggesting thatB7:CD28 interactions represent one of the critical junctures indetermining immune reactivity versus anergy (June C H, Bluestone J A,Linsley P S, Thompson C D: “Role of the CD28 receptor in T-cellactivation.” Immunol Today 15:321, (1994); June C H, Ledbetter J A: “Therole of the CD28 receptor during T-cell responses to antigen.” Annu RevImmunol 11:191, (1993); Schwartz R H: “Co-stimulation of T lymphocytes:The role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production andimmunotherapy.” Cell 71:1065, (1992); Cohen J: “Mounting a targetedstrike on unwanted immune responses” (news; comment). Science 257:751,(1992); Cohen J: “New protein steals the show as ‘co-stimulator’ of Tcells” (news; comment). Science 262:844, (1993)).

In particular, the role of the human B7 antigens, i.e., human B7.1 andB7.2, has been reported to play a co-stimulatory role in T-cellactivation.

1. B7.1 and B7.2 Co-stimulatory Role in T Cell Activation

The elaboration of a successful immune response depends on a series ofspecific interactions between a T cell and an antigen presenting cell.Although the essential first step in this process depends upon thebinding of antigen to the T cell receptor, in the context of the MHCclass II molecule (Lane, P. J. L., F. M. McConnell, G. L. Schieven, E.A. Clark, and J. A. Ledbetter, (1990), “The Role of Class II Moleculesin Human B Cell Activation.” The Journal of Immunology, 144:3684-3692),this interaction alone is not sufficient to induce all the eventsnecessary for a sustained response to a given antigen (Schwartz, R. H.(1990), “A Cell Culture Model for T Lymphocyte Clonal Anergy.” Science,248:1349; Jenkins, M. K. (1992). “The Role of Cell Division in theInduction of Clonal Anergy.” Immunology Today, 13:69; Azuma, M., M.Catabyab, D. Buck, J. H. Phillips, and L. L. Lanier, (1992).“Involvement of CD28 in MHC-unrestricted Cytotoxicity Mediated by aHuman Natural Killer Leukemia Cell Line.” The Journal of Immunology,149:1556-1561; Azuma, M., M. Catabyab, D. Buck, J. H. Phillips, and L.L. Lanier, (1992). “CD28 Interaction with B7 Costimulates PrimaryAllogeneic Proliferative Responses and Cytotoxicity Mediated by SmallResting T Lymphocytes.” J. Exp. Med., 175:353-360).

The involvement of certain other co-stimulatory molecules is necessary(Norton, S. D., L. Zuckerman, K. B. Urdahl, R. Shefner, J. Miller, andM. K. Jenkins. (1992), “The CD28 Ligand, B7, Enhances IL-2 Production byProviding A Costimulatory Signal to T Cells.” The Journal of Immunology,149:1556-1561). “The homodimers CD28 and CTLA-4 expressed on T cells”(June, C. H., J. A. Ledbetter, P. S. Linsley, and C. B. Thompson,(1990), “Role of the CD28 Receptor in T-Cell Activation.” ImmunologyToday, 11:211-216; Linsley, P. S., W. Brady, M. Urnes, L. S. Grosmaire,N. K. Damle, and J. A. Ledbetter, (1991), “CTLA-4 is a Second Receptorfor the B Cell Activation Antigen B7.” J. Exp. Med., 174:561), togetherwith B7.1 (CD80) and B7.2 (CD86) expressed on antigen presenting cells,are major pairs of co-stimulatory molecules necessary for a sustainedimmune response (Azuma, M., H. Yssel, J. H. Phillips, H. Spits, and L.L. Lanier, (1993), “Functional Expression of B7/BB1 on Activated TLymphocytes.” J. Exp. Med., 177:845-850; Freeman, G. J., A. S. Freedman,J. M. Segil, G. Lee, J. F. Whitman, and L M. Nadler, (1989), “B7, A NewMember of the Ig Superfamily with Unique Expression on Activated andNeoplastic B Cells.” The Journal of Immunology, 143:2714-2722; Hathcock,K. S., G. Laslo, H. B. Dickler, J. Bradshaw, P. Linsley, and R. J.Hodes, (1993), “Identification of an Alternative CTLA-4 LigandCostimulatory for T Cell Activation.” Science, 262:905-911; Hart, D. N.J., G. C. Starling, V. L. Calder, and N. S. Fernando, (1993). “B7/BB-1is a Leucocyte Differentiation Antigen on Human Dendritic Cells Inducedby Activation.” Immunology, 79:616-620). It can be shown in vitro thatthe absence of these co-stimulatory signals leads to an aborted T cellactivation pathway and the development of unresponsiveness to thespecific antigen, or anergy. (See, e.g., Harding, F. A., J. G. McArthur,J. A. Gross, D. M. Raulet, and J. P. Allison, (1992). “CD28 MediatedSignalling Co-stimulates Murine T Cells and Prevents Induction of Anergyin T Cell Clones.” Nature, 356:607-609; Gimmi, C.D., G. J. Freeman, J.G. Gribben, G. Gray, and L. M. Nadler, (1993). “Human T-Cell ClonalAnergy is Induced by Antigen Presentation in the Absence of B7Costimulation.” Proc. Natl. Acad. Sci., 90:6586-6590; Tan, P., C.Anasefti, J. A. Hansen, J. Melrose, M. Brunvand, J. Bradshaw, J. A.Ledbetter, and P. S. Linsley, (1993), “Induction of Alloantigen-specificHyporesponsiveness in Human T Lymphocytes by Blocking Interaction ofCD28 with Its Natural Ligand B7/BB1.” J. Exp. Med., 177:165-173).Achievement of in vivo tolerance constitutes a mechanism forimmunosuppression and a viable therapy for organ transplant rejectionand for the treatment of autoimmune diseases. This has been achieved inexperimental models following the administration of CTLA4-Ig (Lenschow,D. J., Y. Zeng, R. J. Thistlethwaite, A. Montag, W. Brady, M. G. Gibson,P. S. Linsley, and J. A. Bluestone, (1992), “Long-Term Survival ofXenogeneic Pancreatic Islet Grafts Induced by CTLA-4Ig.” Science,257:789-795).

The molecules B7.1 and B7.2 can bind to either CD28 or CTLA-4, althoughB7.1 binds to CD28 with a Kd of 200 Nm and to CTLA-4 with a 20-foldhigher affinity (Linsley, P. S., E. A. Clark, and J. A. Ledbetter,(1990), “T-Cell Antigen CD28 Mediates Adhesion with B Cells byInteracting with Activation Antigen B7/BB-1.” Proc. Natl. Acad. Sci.,87:5031-5035; Linsley et al, (1993), “The Role of the CD28 receptorduring T cell responses to antigen,” Annu. Rev. Immunol., 11:191-192;Linesley et al, (1993), “CD28 Engagement by B7/BB-1 Induces TransientDown-Regulation of CD28 Synthesis and Prolonged Unresponsiveness to CD28Signaling,” The Journal of Immunology, 150:3151-3169). B7.2 is expressedon activated B cells and interferon induced monocytes, but not resting Bcells (Freeman, G. J., G. S. Gray, C. D. Gimmi, D. B. Lomarrd, L-J.Zhou, M. White, J. D. Fingeroth, J. G. Gribben, and L M. Nadler, (1991).“Structure, Expression and T Cell Costimulatory Activity of the MurineHomologue of the Human B Lymphocyte Activation Antigen B7,” J. Exp.Med., 174:625-631). B7.2, on the other hand, is constitutively expressedat very low levels on resting monocytes, dendritic cells and B cells,and its expression is enhanced on activated T cells, NK cells and Blymphocytes (Azuma, M. D. Ito, H. Yagita, K. Okumura, J. H. Phillips, L.L. Lanier, and C. Somoza, “1993”, “IB70 Antigen is a Second Ligand forCTLA-4 and CD28, ” Nature, 366:76-79). Although B7.1 and B7.2 can beexpressed on the same cell type, their expression on B cells occurs withdifferent kinetics (Lenschow, D. J., G. H. Su, L. A. Zuckerman, N.Nabavi, C. L. Jellis, G. S. Gray, J. Miller, and J. A. Bluestone,(1993), “Expression and Functional Significance of an Additional Ligandfor CTLA-4,” Proc. Natl. Acad. Sci., USA, 90:11054-11058; Boussiotis, V.A., G. J. Freeman, J. G. Gribben, J. Daley, G. Gray, and L. M. Nadler,(1993), “Activated Human B Lymphocytes Express Three CTLA-4Counter-receptors that Co-stimulate T-Cell Activation,” Proc. Natl.Acad. Sci., USA, 90:11059-11063). Further analysis at the RNA level hasdemonstrated that B7.2 mRNA is constitutively expressed, whereas B7.1mRNA is detected 4 hours after activation and initial low levels of B7.1protein are not detectable until 24 hours after stimulation (Boussiotis,V. A., G. J. Freeman, J. G. Gribben, J. Daley, G. Gray, and L. M.Nadler, (1993), “Activated Human B Lymphocytes Express Three CTLA-4Counter-receptors that Co-stimulate T-Cell Activation,” Proc. Natl.Acad. Sci., USA, 90:11059-11063). CTLA-4/CD28 counter receptors,therefore, may be expressed at various times after B Cell activation.

The differential temporal expression of B7.1 and B7.2 suggests that theinteraction of these two molecules with CTLA-4 and/or CD28 deliverdistinct but related signals to the T cell (LaSalle, J. M., P. J.Tolentino, G. J. Freeman, L. M. Nadler, and D. A. Hafler, (1992), “CD28and T Cell Antigen Receptor Signal Transduction Coordinately RegulateIntedeukin 2 Gene Expression In Response to Superantigen Stimulation,”J. Exp. Med., 176:177-186; Vandenberghe, P., G. J. Freeman, L. M.Nadler, M. C. Fletcher, M. Kamoun, L. A. Turka, J. A. Ledbetter, C. B.Thompson, and C. H. June, (1992), “Antibody and B7/BB1-mediated Ligationof the CD28 Receptor Induces Tyrosine Phosphorylation in Human T Cells,”The Journal of Experimental Medicine, 175:951-960). The exact signalingfunctions of CTLA-4 and CD28 on the T cell are currently unknown(Janeway, C. A., Jr. and K. Bottomly, (1994), “Signals and Signs forLymphocyte Responses,” Cell, 76.275285). However, it is possible thatone set of receptors could provide the initial stimulus for T cellactivation and the second, a sustained signal to allow furtherelaboration of the pathway and clonal expansion to take place (Linsley,P. S., J. L. Greene, P. Tan, J. Bradshaw, J. A. Ledbetter, C. Anasetti,and N. K. Damle, (1992), “Coexpression and Functional Cooperation ofCTLA-4 and CD28 on Activated T Lymphocytes,” J. Exp. Med.,176:1595-1604). The current data supports the two-signal hypothesisproposed by Jenkins and Schwartz (Schwartz, R. H., (1990), “A CellCulture Model for T Lymphocyte Clonal Anergy,” Science, 248:1349;Jenkins, M. K., (1992), “The Role of Cell Division in the Induction ofClonal Anergy,” Immunology Today, 13:69) that both a TCR andco-stimulatory signal are necessary for T cell expansion, lymphokinesecretion and the full development of effector function (Greenan, V. andG. Kroemer, (1993), “Multiple Ways to Cellular Immune Tolerance,”Immunology Today, 14:573). The failure to deliver the second signalresults in the inability of T cells to secrete IL-2 and renders the cellunresponsive to antigen.

Structurally, both B7.1 and B7.2 contain extracellular immunoglobulinsuperfamily V and C-like domains, a hydrophobic transmembrane region anda cytoplasmic tail (Freeman, G. J., J. G. Gribben, V. A. Boussiotis, J.W. Ng, V. Restivo, Jr., L. A. Lombard, G. S. Gray, and L. M. Nadler,(1993), “Cloning of B7-2: A CTLA-4 Counter-receptor that Co-stimulatesHuman T Cell Proliferation,” Science, 262:909). Both B7.1 and B7.2 areheavily glycosylated. B7.1 is a 44-54 kD glycoprotein comprised of a 223amino acid extracellular domain, a 23 amino acid transmembrane domain,and a 61 amino acid cytoplasmic tail. B7.1 contains 3 potential proteinkinase phosphorylation sites. (Azuma, M., H. Yssel, J. H. Phillips, H.Spits, and L. L. Lanier, (1993), “Functional Expression of B7/BB1 onActivated T Lymphocytes,” J. Exp. Med., 177:845-850). B7.2 is a 306amino acid membrane glycoprotein. It consists of a 220 amino acidextracellular region, a 23 amino acid hydrophobic transmembrane domainand a 60 amino acid cytoplasmic tail (Freeman, G. J., A. S. Freedman, J.M. Segil, G. Lee, J. F. Whitman, and L M. Nadler, (1989), “B7, A NewMember of the Ig Superfamily with Unique Expression on Activated andNeoplastic B Cells,” The Journal of Immunology, 143:2714-2722). Althoughboth B7.1 and B7.2 genes are localized in the same chromosomal region(Freeman, G. J., D. B. Lombard, C. D. Gimmi, S. A. Brod, L Lee, J. C.Laning, D. A. Hafler, M. E. Dorf, G. S. Gray, H. Reiser, C. H. June, C.B. Thompson, and L. M. Nadler, (1992), “CTLA-4 and CD28 mRNA areCoexpressed in Most T Cells After Activation,” The Journal ofImmunology, 149:3795-3801; Schwartz, R. H., (1992), “Costimulation of TLymphocytes: The Role of CD28, CTLA-4, and B7/BB1” in Selvakumar, A., B.K. Mohanraj, R. L. Eddy, T. B. Shows, P. C. White, C. Perrin, and B.Dupont, (1992), “Genomic Organization and Chromosomal Location of theHuman Gene Encoding the B-Lymphocyte Activation Antigen B7,”Immunogenetics, 36:175-181), these antigens do not share a high level ofhomology. The overall homology between B7.1 and B7.2 is 26% and betweenmurine B7.1 and human S7 is 27% (Azuma, M., H. Yssel, J. H. Phillips, H.Spits, and L. L. Lanier, (1993), “Functional Expression of B7/BB1 onActivated T Lymphocytes,” J. Exp. Med., 177:845-850; Freeman, G. J., A.S. Freedman, J. M. Segil, G. Lee, J. F. Whitman, and L M. Nadler,(1989), “B7, A New Member of the Ig Superfamily with Unique Expressionon Activated and Neoplastic B Cells,” The Journal of Immunology,143:2714-2722). Although alignment of human B7.1 human B7.2 and murineB.1 sequences shows few stretches of lengthy homology, it is known thatall three molecules bind to human CTLA-4 and CD28. Thus, there is mostlikely a common, or closely homologous region shared by the threemolecules that may be either contiguous or conformational. This regionmay constitute the binding site of the B7.1 and B7.2 molecules to theircounter-receptors. Antibodies raised against these epitopes couldpotentially inhibit the interaction of B7 with its counter-receptor onthe T cell. Furthermore, antibodies that cross-reacted with this regionon both B7.1 and 37.2 molecules would potentially have practicaladvantages over antibodies directed against B7.1 or B7.2 separately.

2. Blockade of the B7/CD28 Interaction

Blocking of the B7/CD28 interaction offers the possibility of inducingspecific immunosuppression, with potential for generating long lastingantigen-specific therapeutic effects. Antibodies to either B7.1 or B7.2have been shown to block T cell activation, as measured by theinhibition of IL-2 production in vitro (DeBoer, M., P. Parren, J. Dove,F. Ossendorp, G. van der Horst, and J. Reeder, (1992), “FunctionalCharacterization of a Novel Anti-B7 Monoclonal Antibody,” Eur. Journalof Immunology, 22:3071-3075; Azuma, M., H. Yssel, J. H. Phillips, H.Spits, and L. L. Lanier, (1993), “Functional Expression of B7/BB1 onActivated T Lymphocytes,” J. Exp. Med., 177:845-850). However, differentantibodies have been shown to vary in their immunosuppressive potency,which may reflect either their affinity or epitope specificity.CTLA-4/lg fusion protein and anti-CD28 Fabs were shown to have similareffects on the down regulation of IL-2 production.

In vivo administration of a soluble CTLA-4/lg fusion protein has beenshown to suppress T cell—dependent antibody responses in mice (Linsley,P. S., J. L. Greene, P. Tan, J. Bradshaw, J. A. Ledbetter, C. Anasetti,and N. K. Damle, (1992), “Coexpression and Functional Cooperation ofCTLA-4 and CD28 on Activated T Lymphocytes,” J. Exp. Med.,176:1595-1604; Lin, H., S. F. Builing, P. S. Linsley, R. O. Wei, C. D.Thompson, and L. A. Turka, (1993), “Long-term Acceptance of MajorHistocompatibility Complex Mismatched Cardiac Allografts Induced byCTLA-4-Ig Plus Donor Specific Transfusion,” J. Exp. Med., 178:1801) and,furthermore, larger doses were also able to suppress responses to asecond immunization, demonstrating the feasibility of this approach forthe treatment of antibody mediated autoimmune disease. In addition,CTLA-4/Ig was able to prevent pancreatic islet cell rejection in mice bydirectly inhibiting the interaction of T cells and B7.1/B7.2 antigenpresenting cells (Lenschow, D. J., G. H. Su, L. A. Zuckerman, N. Nabavi,C. L. Jellis, G. S. Gray, J. Miller, and J. A. Bluestone, (1993),“Expression and Functional Significance of an Additional Ligand forCTLA-4,” Proc. Natl. Acad. Sci., USA, 90:11054-11058). In this case,long term donor specific tolerance was achieved.

3. Recombinant Phage Display Technology for Antibody Selection

To date, no monoclonal antibodies which cross-react with both B7.1 andB7.2 have been reported. As noted, such antibodies would potentially behighly desirable as immunosuppressants. Phage display technology isbeginning to replace traditional methods for isolating antibodiesgenerated during the immune response, because a much greater percentageof the immune repertoire can be assessed than is possible usingtraditional methods. This is in part due to PEG fusion inefficiency,chromosomal instability, and the large amount of tissue culture andscreening associated with heterohybridoma production. Phage displaytechnology, by contrast, relies on molecular techniques for potentiallycapturing the entire repertoire of immunoglobulin genes associated withthe response to a given antigen.

This technique is described by Barber et al, Proc. Natl. Acad. Sci.,USA, 88, 7978-7982, (1991). Essentially, immunoglobulin heavy chaingenes are PCR amplified and cloned into a vector containing the geneencoding the minor coat protein of the filamentous phage M13 in such away that a heavy chain fusion protein is created. The heavy chain fusionprotein is incorporated into the M13 phage particle together with thelight chain genes as it assembles. Each recombinant phage contains,within its genome, the genes for a different antibody Fab molecule whichit displays on its surface. Within these libraries, in excess of 10⁶different antibodies can be cloned and displayed. The phage library ispanned on antigen coated microliter wells, non-specific phage are washedoff, and antigen binding phage are eluted. The genome from theantigen-specific clones is isolated and the gene III is excised, so thatantibody can be expressed in soluble Fab form for furthercharacterization. Once a single Fab is selected as a potentialtherapeutic candidate, it may easily be converted to a whole antibody. Apreviously described expression system for converting Fab sequences towhole antibodies is IDEC's mammalian expression vector NEOSPLA. Thisvector contains either human gamma 1 or gamma 4 constant region genes.CHO cells are transfected with the NEOSPLA vectors and afteramplification this vector system has been reported to provide very highexpression levels (>30 pg/cell/day) can be achieved.

4. Primatized Antibodies

Another highly efficient means for generating recombinant antibodies isdisclosed by Newman, (1992), Biotechnology, 10, 1455-1460. Moreparticularly, this technique results in the generation of primatizedantibodies which contain monkey variable domains and human constantsequences. This reference is incorporated by reference in its entiretyherein. Moreover, this technique is also described in commonly assignedU.S. application Ser. No. 08/379,072, filed on Jan. 25, 1995, which is acontinuation of U.S. Ser. No. 07/912,292, filed Jul. 10, 1992, which isa continuation-in-part of U.S. Ser. No. 07/856,281, filed Mar. 23, 1992,which is finally a continuation-in-part of U.S. Ser. No. 07/735,064,filed Jul. 25, 1991. Ser. No. 08/379,072 and the parent applicationthereof are incorporated by reference in their entirety herein.

This technique modifies antibodies such that they are not antigenicallyrejected upon administration in humans. This technique relies onimmunization of cynomolgus monkeys with human antigens or receptors.This technique was developed to create high affinity monoclonalantibodies directed to human cell surface antigens.

Antibodies generated in this manner have previously been reported todisplay human effector function, have reduced immunogenicity, and longserum half-life. The technology relies on the fact that despite the factthat cynomolgus monkeys are phylogenetically similar to humans, theystill recognize many human proteins as foreign and therefore mount animmune response. Moreover, because the cynomolgus monkeys arephylogenetically close to humans, the antibodies generated in thesemonkeys have been discovered to have a high degree of amino acidhomology to those produced in humans. Indeed, after sequencing macaqueimmunoglobulin light and heavy chain variable region genes, it was foundthat the sequence of each gene family was 85-98% homologous to its humancounterpart (Newman et al, (1992), Id.). The first antibody generated inthis way, an anti-CD4 antibody, was 91-92% homologous to the consensussequence of human immunoglobulin framework regions. Newman et al,Biotechnology, 10:1458-1460, (1992).

Monoclonal antibodies specific to the human B7 antigen have beenpreviously described in the literature. For example, Weyl et al, Hum.Immunol., 31(4), 271-276, (1991) describe epitope mapping of humanmonoclonal antibodies against HLA-B-27 using natural and mutatedantigenic variants. Also, Toubert et al, Clin. Exp. Immunol., 82(1),16-20, (1990) describe epitope mapping of an HLA-B27 monoclonal antibodythat also reacts with a 35-KD bacterial outer membrane protein. Also,Valle et al, Immunol., 69(4), 531-535, (1990) describe a monoclonalantibody of the IgG1 subclass which recognizes the B7 antigen expressedin activated B cells and HTLV-1-transformed T cells. Further, Toubert etal, J. Immunol., 141(7), 2503-9, (1988) describe epitope mapping ofHLA-B27 and HLA-B7 antigens using intradomain recombinants constructedby making hybrid genes between these two alleles in E. coli.

High expression of B7 antigen has been correlated to autoimmune diseasesby some researchers. For example, Ionesco-Tirgoviste et al, Med.Interre, 24(1), 11-17, (1986) report increased B7 antigen expression intype 1 insulin-dependent diabetes. Also, the involvement of B7 antigenexpression on dermal dendritic cells obtained from psoriasis patientshas been reported. (Nestle et al, J. Clin. Invest., 94(1), 202-209,(1994)).

Further, the inhibition of anti-HLA-B7 alloreactive CTL usingaffinity-purified soluble HLA-B7 has been reported in the literature.(Zavazava et al, Transplantation, 51(4), 838-42, (1991)). Further, theuse of B7 receptor soluble ligand, CTLA-4-Ig to block B7 activity (See,e.g., Lenschow et al, Science, 257, 789, 7955 (1992)) in animal modelsand a B7-1-Ig fusion protein capable of inhibiting B7 has been reported.

SUMMARY AND OBJECTS OF THE INVENTION

An object of the invention is to produce and identify novel macaqueantibodies to human B7 antigen, more specifically to human B7.1 antigenand/or human B7.2 antigen.

More specifically, it is an object of the present invention to produceand identify novel macaque antibodies to human B7 antigen, i.e., humanB7.1 and human B7.2 antigen by screening of phage display librariesand/or monkey heterohybridomas using B lymphocytes obtained from humanB7 antigen, i.e., human B7.1 or B7.2 antigen immunized monkeys.

It is another specific object of the invention to provide anti-B7 monkeymonoclonal antibodies and primatized forms thereof which specificallybind human B7.1 and/or B7.2 antigen which inhibit the B7/CD86 pathwayand B7 stimulation of activated T cells, thereby inhibiting IL-2production and T cell proliferation and functioning as effectiveimmunosuppressants.

It is another object of the invention to provide anti-human B7.1 andanti-human B7.2 monkey monoclonal antibodies and primatized formsthereof which inhibit antigen driven responses in donor spleen cellcultures, e.g., antigen specific IgG responses, IL-2 production and cellproliferation.

It is another specific object of the invention to identify particularmonkey monoclonal antibodies specific to human B7.1 and human B7.2antigen and primatized forms thereof having advantageous properties,i.e., affinity, immunosuppressive activity, which are useful astherapeutics. More specifically, these monkey antibodies and primatizedforms thereof are to be used, e.g., as immunosuppressants, i.e., toblock antigen driven immune responses, to treat autoimmune diseases suchas psoriasis, rheumatoid arthritis, systemic erythematosus (SLE), type 1diabetes mellitus, idiopathic thrombocytopenia purpura (ITP), and toprevent organ rejection.

It is another object of the invention to provide pharmaceuticalcompositions containing one or more monkey monoclonal antibodiesspecific to human B7 antigen, i.e., human B7.1 and/or human B7.2antigen, or primatized forms thereof, and a pharmaceutically acceptablecarrier or excipient. These compositions will be used, e.g., asimmunosuppressants to treat autoimmune diseases, e.g., idiopathicthrombocytopenia purpura (ITP) and systemic lupus erythematosus (SLE),to block antigen driven immune responses, and to prevent organ rejectionin transplant recipients.

It is another object of the invention to provide novel methods oftherapy by administration of therapeutically effective amounts of one ormore monkey or primatized monoclonal antibodies which specifically bindto B7 antigen, i.e., human B7.1 and/or B7.2 antigens. Such therapeuticmethods are useful for treatment of diseases treatable by inhibition ofthe B7:CD28 pathway e.g., autoimmune diseases such as idiopathicthrombocytopenia purpura (ITP), systemic lupus erythematosus (SLE), type1 diabetes mellitus, psoriasis, rheumatoid arthritis, multiplesclerosis, aplastic anemia, as well as for preventing rejection intransplantation subjects.

It is still another object of the invention to provide transfectants,e.g., CHO cells, which express at least the variable heavy and lightdomains of monkey monoclonal antibodies specific to the human B7.1and/or B7.2 antigen.

It is another object of the invention to provide nucleic acid sequenceswhich encode the variable heavy and/or light domains of monkeymonoclonal antibodies specific to human B7.1 and/or human B7.2 antigen,and expression vectors which provide for the expression of primatizedantibodies containing these nucleic acid sequences.

Definitions

The following terms are defined so that the invention may be moreclearly understood.

Depleting antibody—an antibody which kills activated B cells or otherantigen presenting cells.

Non-depleting antibody—an antibody which blocks the co-stimulatoryaction of B7 and T cell activating ligands CD28 and CTLA-4. Thus, itanergizes but does not eliminate the antigen presenting cell.

Primatized antibody—a recombinant antibody which has been engineered tocontain the variable heavy and light domains of a monkey antibody, inparticular, a cynomolgus monkey antibody, and which contains humanconstant domain sequences, preferably the human immunoglobulin gamma 1or gamma 4 constant domain (or PE variant). The preparation of suchantibodies is described in Newman et al, (1992), “Primatization ofRecombinant Antibodies for Immunotherapy of Human Diseases: AMacaque/Human Chimeric Antibody Against Human CDH, Biotechnology,10:1458-1460; also in commonly assigned Ser No. 08/379,072 both of whichare incorporated by reference in their entirety herein. These antibodieshave been reported to exhibit a high degree of homology to humanantibodies, i.e., 85-98%, display human effector functions, have reducedimmunogenicity, and may exhibit high affinity to human antigens.B7 antigens—B7 antigens in this application include, e.g., human B7,B7.1 and B7.2 antigens. These antigens bind to CD28 and/or CTLA-4. Theseantigens have a co-stimulatory role in T cell activation. Also, these B7antigens all contain extracellular immunoglobulin superfamily V andC-like domains, a hydrophobic transmembrane region and a cytoplasmictail. (See, Freeman et al, Science, 262:909, (1993)), and are heavilyglycosylated.Anti-B7 antibodies—Antibodies, preferably monkey monoclonal antibodiesor primatized forms thereof, which specifically bind human B7 antigens,e.g., human B7.1 and/or B7.2 antigen with a sufficient affinity to blockthe B7:CD28 interaction and thereby induce immunosuppression.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the pMS vector used to screen recombinant immunoglobulinlibraries produced against B7 displayed on the surface of filamentousphage which contains primers based on macaque immunoglobulin sequences.

FIG. 2 depicts the NEOSPLA expression vector used to express the subjectprimatized antibodies specific to human B7.1 antigen.

FIG. 3 depicts monkey serum anti-B7.1 titers directed against cellsurface B7.1 on transfected CHO cells.

FIG. 4 depicts inhibition of radiolabeled sB7.1 binding by SB7.1affinity-purified monkey antibodies in the presence of unlabeled SB7 andMab L307.4 murine anti-B7.1.

FIG. 5 depicts inhibition of binding of radiolabeled monkey 135 andL3707.4 anti-B7.1 antibodies to B7 positive human SB cells bycompetition with affinity-purified SB7.1.

FIG. 6 depicts inhibition of radiolabeled B7-Ig binding to activatedhuman peripheral blood T cells by competing with unlabeled SB7.1 murineanti-B7.1 (L307.4) and monkey 1127 affinity purified serum antibodies.

FIG. 7 depicts inhibition of IL-2 protein in mixed lymphocyte culturesby anti-B7.1 affinity-purified monkey serum antibodies.

FIG. 8 a (SEQ ID NOS: 1-2) depicts the amino acid and nucleic acidsequence of a primatized form of the light chain of 7C10.

FIG. 8 b (SEQ ID NOS: 3-4) depicts the amino acid and nucleic acidsequence of a primatized farm of the heavy chain of 7C10.

FIG. 9 a (SEQ ID NOS: 5-6) depicts the amino acid and nucleic acidsequence of a primatized form of the light chain of 7B6.

FIG. 9 b (SEQ ID NOS: 7-8) depicts the amino acid and nucleic acidsequence of a primatized form of the heavy chain of 7B6.

FIG. 10 a (SEQ ID NOS: 9-10) depicts the amino acid and nucleic acidsequence of a primatized light chain 16C10.

FIG. 10 b (SEQ ID NOS:11-12) depicts the amino acid and nucleic acidsequence of a primatized heavy chain 16C10.

DETAILED DESCRIPTION OF THE INVENTION

As described above, the present invention relates to the manufacture ofnovel monkey monoclonal antibodies which specifically bind human B7.1and/or human B7.2 antigen, as well as primatized antibodies derivedtherefrom. These antibodies possess high affinity to human B7.1 and/orB7.2 and therefore may be used as immunosuppressants which inhibit theB7:CD86 pathway.

Preparation of monkey monoclonal antibodies will preferably be effectedby screening of phage display libraries or by preparation of monkeyheterohybridomas using B lymphocytes obtained from B7 (e.g., human B7.1and/or B7.2) immunized monkeys.

As noted, the first method for generating anti-B7 antibodies involvesrecombinant phage display technology. This technique is generallydescribed supra.

Essentially, this will comprise synthesis of recombinant immunoglobulinlibraries against B7 antigen displayed on the surface of filamentousphage and selection of phage which secrete antibodies having highaffinity to B7.1 and/or B7.2 antigen. As noted supra, preferablyantibodies will be selected which bind to both human B7.1 and B7.2. Toeffect such methodology, the present inventors have created a uniquelibrary for monkey libraries which reduces the possibility ofrecombination and improves stability. This vector, pMS, is described indetail infra, and is shown in FIG. 1.

Essentially, to adopt phage display for use with macaque libraries, thisvector contains specific primers for PCR amplifying monkeyimmunoglobulin genes. These primers are based on macaque sequencesobtained while developing the primatized technology and databasescontaining human sequences.

Suitable primers are disclosed in commonly assigned Ser. No. 08/379,072incorporated by reference herein.

The second method involves the immunization of monkeys, i.e., macaques,against human B7 antigen, preferably against human B7.1 and B7.2antigen. The inherent advantage of macaques for generation of monoclonalantibodies is discussed supra. In particular, such monkeys, i.e.,cynomolgus monkeys, may be immunized against human antigens orreceptors. Moreover, the resultant antibodies may be used to makeprimatized antibodies according to the methodology of Newman et al,Biotechnology, 10, 1455-1460, (1992), and Newman et al, commonlyassigned U.S. Ser. No. 08/379,072, filed Jan. 25, 1995, which areincorporated by reference in their entirety.

The significant advantage of antibodies obtained from cynomolgus monkeysis that these monkeys recognize many human proteins as foreign andthereby provide for the formation of antibodies, some with high affinityto desired human antigens, e.g., human surface proteins and cellreceptors. Moreover, because they are phylogenetically close to humans,the resultant antibodies exhibit a high degree of amino acid homology tothose produced in humans. As noted above, after sequencing macaqueimmunoglobulin light and heavy variable region genes, it was found thatthe sequence of each gene family was 85-88% homologous to its humancounterpart (Newman et al, (1992), Id.).

Essentially, cynomolgus macaque monkeys are administered human B7antigen, e.g., human B7.1 and/or human B7.2 antigen, B cells areisolated therefrom, e.g., lymph node biopsies are taken from theanimals, and B lymphocytes are then fused with KH6/B5 (mouse×human)heteromyeloma cells using polyethylene glycol (PEG). Heterohybridomassecreting antibodies which bind human B7 antigen, e.g., human B7.1and/or human B7.2 antigen, are then identified.

Antibodies which bind to both B7.1 and B7.2 are desirable because suchantibodies potentially may be used to inhibit the interaction of B7.1and B7.2, as well as B7 with their counter-receptors, i.e., human CTLA-4and CD28. Antibodies against these epitopes may inhibit the interactionof both human B7.1 and human B7.2 with their counter receptors on the Tcell. This may potentially provide synergistic effects.

However, antibodies which bind to only one of human B7 antigen, B7.1antigen or B7.2 antigen, are also highly desirable because of theco-involvement of these molecules in T cell activation, clonal expansionlymphokine (IL-2) secretion, and responsiveness to antigen. Given thatboth human B7.1 and B7.2 bind to human CTLA-4 and CD28, it is probablethat there is at least one common or homologous region (perhaps a sharedconformational epitope or epitopes) to which macaque antibodies maypotentially be raised.

The present inventors elected to immunize macaques against human B7.1antigen using recombinant soluble B7.1 antigen produced in CHO cells andpurified by affinity chromatography using a L307.4-sepharose affinitycolumn. However, the particular source of human B7 antigen, human B7.1antigen or human B7.2 antigen is not critical, provided that it is ofsufficient purity to result in a specific antibody response to theparticular administered B7 antigen and potentially to other B7 antigens.

The human B7 antigen, human B7.1 antigen (also called CD80) and humanB7.2 antigen (also called CD86) genes have been cloned, and sequenced,and therefore may readily be manufactured by recombinant methods.

Preferably, the administered human B7 antigen, human B7.1 antigen and/orhuman B7.2 antigen will be administered in soluble form, e.g., byexpression of a B7, B7.1 or B7.2 gene which has its transmembrane andcytoplasmic domains removed, thereby leaving only the extracellularportion, i.e., the extracellular superfamily V and C-like domains. (See,e.g., Grumet et al, Hum. Immunol., 40(3), p. 228-234, 1994, whichteaches expression of a soluble form of human B7, which is incorporatedby reference in its entirety herein).

The macaques will be immunized with the B7, B7.1 and/or B7.2 antigen,preferably a soluble form thereof, under conditions which result in theproduction of antibodies specific thereto. Preferably, the soluble humanB7, B7.1 or B7.2 antigen will be administered in combination with anadjuvant, e.g., Complete Freund's Adjuvant (CFA), Alum, Saponin, orother known adjuvants, as well as combinations thereof. In general, thiswill require repeated immunization, e.g., by repeated injection, overseveral months. For example, administration of soluble B7.1 antigen waseffected in adjuvant, with booster immunizations, over a 3 to 4 monthperiod, with resultant production of serum containing antibodies whichbound human B7.1 antigen.

After immunization B cells are collected, e.g., by lymph node biopsiestaken from the immunized animals and B lymphocytes fused with KH6/B5(mouse×human) heteromyeloma cells using polyethylene glycol. Methods forpreparation of such heteromyelomas are known and may be found in U.S.Ser. No. 08/379,072 by Newman et al, filed on Jan. 25, 1995 andincorporated by reference herein.

Heterohybridomas which secrete antibodies which bind human B7, B7.1and/or B7.2 are then identified. This may be effected by knowntechniques. For example, this may be determined by ELISA orradioimmunoassay using enzyme or radionuclide labelled human B7, B7.1and/or B7.2 antigen.

Cell lines which secrete antibodies having the desired specificity tohuman B7, B7.1 and/or B7.2 antigen are then subcloned to monoclonality.

In the present invention, the inventors screened purified antibodies fortheir ability to bind to soluble B7.1 antigen coated plates in an ELISAassay, antigen positive B cells, and CHO transfectomas which expresshuman B7.1 antigen on their cell surface. In addition, the antibodieswere screened for their ability to block B cell/T cell interactions asmeasured by IL-2 production and tritiated thymidine uptake in a mixedlymphocyte reaction (MLR), with B7 binding being detected using¹²⁵I-radiolabeled soluble B7.1 (SB7.1).

Also, affinity purified antibodies from macaques were tested for theirreactivity against CHO transfectants which expressed B7.1/Ig fusionproteins, and against CHO cells which produced human B7.2 antigen. Theseresults indicated that the B7.1 immune sera bound to the B7.2transfectomas. Binding of antibodies to B7.2 antigen may be confirmedusing soluble B7.2-Ig reagents. As discussed in the examples, this maybe effected by producing and purifying B7.2-Ig from CHO transfectomas insufficient quantities to prepare a B7.2-Ig-sepharose affinity column.Those antibodies which cross-react with B7.2 will bind theB7.2-Ig-sepharose column.

Cell lines which express antibodies which specifically bind to human B7antigen, B7.1 antigen and/or B7.2 antigen are then used to clonevariable domain sequences for the manufacture of primatized antibodiesessentially as described in Newman et al, (1992), Id. and Newman et al,U.S. Ser. No. 379,072, filed Jan. 25, 1995, both of which areincorporated by reference herein. Essentially, this entails extractionof RNA therefrom, conversion to cDNA, and amplification thereof by PCRusing Ig specific primers. Suitable primers are described in Newman etal, 1992, Id. and in U.S. Ser. No. 379,072. (See, in particular, FIG. 1of U.S. Ser. No. 379,072).

The cloned monkey variable genes are then inserted into an expressionvector which contains human heavy and light chain constant region genes.Preferably, this is effected using a proprietary expression vector ofIDEC, Inc., referred to as NEOSPLA. This vector is shown in FIG. 2 andcontains the cytomegalovirus promoter/enhancer, the mouse beta globinmajor promoter, the SV40 origin of replication, the bovine growthhormone polyadenylation sequence, neomycin phosphotransferase exon 1 andexon 2, human immunoglobulin kappa or lambda constant region, thedihydrofolate reductase gene, the human immunoglobulin gamma 1 or gamma4 PE constant region and leader sequence. This vector has been found toresult in very high level expression of primatized antibodies uponincorporation of monkey variable region genes, transfection in CHOcells, followed by selection in G418 containing medium and methotrexateamplification.

For example, this expression system has been previously disclosed toresult in primatized antibodies having high avidity (Kd≦10⁻¹⁰ M) againstCD4 and other human cell surface receptors. Moreover, the antibodieshave been found to exhibit the same affinity, specificity and functionalactivity as the original monkey antibody. This vector system issubstantially disclosed in commonly assigned U.S. Ser. No. 379,072,incorporated by reference herein as well as U.S. Ser. No. 08/149,099,filed on Nov. 3, 1993, also incorporated by reference in its entiretyherein. This system provides for high expression levels, i.e., >30pg/cell/day.

As discussed infra, the subject inventors have selected four leadcandidate monkey monoclonal antibodies which specifically bind the B7.1antigen, and which may also bind the B7.2 antigen. These monkeymonoclonal antibodies are referred to herein as 7B6, 16C10, 7C10 and20C9.

As discussed in greater detail infra, these antibodies were evaluatedfor their ability to block B cell/T cell interactions as measured byIL-2 production and tritiated thymidine uptake in a mixed lymphocytereaction for T cell binding experiments for T cell binding, human bodycoat peripheral blood lymphocytes were cultured for 3-6 days in thepresence of PHA stimulator. B7 binding was radioassayed using¹²⁵I-radiolabeled soluble B7.1. The observed results indicate that allof these antibodies bind B7.1 antigen with high affinity and effectivelyblock 2 cell/T cell interactions as evidenced by reduced IL-2 productionand reduced proliferation of mixed lymphocyte cultures.

The properties of these particular monkey monoclonal antibodies aresummarized below:

1. To demonstrate the monkey antibodies' ability to block the physicalinteraction between CTLA4-Ig, varying concentrations of the monkeyanti-B7.1 antibodies and unlabeled CTLA4-IG were incubated withradiolabeled CTLA4-Ig^(I125). The results of the inhibition assay showedthat the IC50 (the concentration of inhibitor which results in 50%inhibition) for the monkey antibodies are:

a: 7C10: 0.39 μg/Ml b: 16C10: 1.60 μg/Ml c: 20C9: 3.90 μg/Ml d: 7B6:39.0 μg/Ml

2. Scatchard analysis showed that the apparent affinity constants (Kd)for the monkey antibodies binding to B7-Ig coated plates wereapproximated to be:

a: 7C10:  6.2 × 10⁻⁹M b: 16C10:  8.1 × 10⁻⁹M c: 7B6: 10.7 × 10⁻⁹M d:20C9: 16.8 × 10⁻⁹M

3. The antibodies were tested in vitro in a mixed lymphocyte reactionassay (MLR). The MLR showed that all 4 anti-B7.1 antibodies inhibit IL-2production to different extents as shown by the following Ibgo values:

a: 7B6:  5.0 μg/M b: 16C10: <0.1 μg/M c: 20C9:  2.0 μg/M d: 7C10:  5.0μg/M

4. The monkey anti-B7.1 antibodies were tested for their ability to bindB7 on human peripheral blood lymphocytes (PBL). FACS analysis showedthat all 4 monkey antibodies tested positive.

5. Monkey antibodies 16C10, 7B6, 7C10 and 20C9 were tested for C1qbinding by FACS analysis. Results showed 7C10 monkey Ig had strong humanC1q binding after incubating with B7.1 CHO-transfected cells. 16C10 waspositive, while 20C9 and 7B6 monkey antibodies were negative.

6. To select an animal model for path-tox studies, the monkey antibodieswere tested with animal blood from different species. It was determinedthat the monkey anti-B7.1 antibodies cross-reacted with human,chimpanzee, and possibly baboon.

Based on these properties, it would appear that three monkey monoclonalantibodies possess the most advantageous properties, 16C10, 7C10 and20C9, with 16C10 and 7C10 being somewhat better than 20C9.

Using the techniques described supra, and in commonly assigned U.S. Ser.No. 08/379,072, the present inventors have cloned the variable domainsof 7C10, 7B6 and 16C10, and provide the amino acid and nucleic acidsequences of primatized forms of the 7C10 light chain, 7C10 heavy chain,7B6 light chain, 7B6 heavy chain, 16C10 light chain and 16C10 heavychain. These amino acid and nucleic acid sequences may be found in FIGS.8 a and 8 b, 9 a and 9 b, and 10 a and 10 b. The DNA and amino acidsequence for the human gamma 1 constant domain may be found in U.S. Ser.No. 08/379,072.

As discussed supra, these primatized antibodies are preferably expressedusing the NEOSPLA expression vector shown in FIG. 2 which issubstantially described in commonly assigned Ser. Nos. 08/379,072 and08/149,099, both of which applications are incorporated by referenceherein.

As previously noted, the subject primatized antibodies will preferablycontain either the human immunoglobulin gamma 1 or gamma 4 constantregion, with gamma 4 preferably mutated at two positions to create gamma4 PE. The gamma 4 PE mutant contains two mutations, a glutamic acid inthe CH2 region introduced to eliminate residual FCR binding, and aproline substitution in the hinge region, intended to enhance thestability of the heavy chain disulfide bond interaction. (See, Alegre etal, J. Immunol., 148, 3461-3468, (1992); and Angel et al, Mol. Immunol.,30, 105-158, (1993), both of which are incorporated by referenceherein).

Whether the subject primatized antibodies contain the gamma 1, gamma 4or gamma 4 PE constant region largely depends on the particular diseasetarget. Preferably, depleting and non-depleting primatized IgG1 and IgG4antibodies are created and tested against specific disease targets.

Given the described binding and functional properties of the subjectmonkey monoclonal antibodies, these anti-B7.1 monoclonal antibodies andprimatized forms thereof should be well suited as therapeutic agents forblocking the B7:CD28 interaction thereby providing forimmunosuppression. In particular, given their high affinity to B7.1antigen and ability to block B cell/T cell interactions as measured byIL-2 production and tritiated thymidine uptake in mixed lymphocyteculture as well as their ability to effectively inhibit antigen drivenresponses in donor spleen cell cultures as shown by reduced antigenspecific IgG responses, IL-2 production and cell proliferation, thesemonkey monoclonal antibodies and primatized forms thereof shouldfunction as effective immunosuppressants which modulate the B7:CD28pathway. This is significant for the treatment of many diseases whereinimmunosuppression is therapeutically desirable, e.g., autoimmunediseases, to inhibit undesirable antigen specific IgG responses, andalso for prevention of organ rejection and graft-versus-host disease.Essentially, the subject antibodies will be useful in treating anydisease wherein suppression of the B7:CD28 pathway is therapeuticallydesirable.

Key therapeutic indications for the subject anti-B7.1 antibodiesinclude, by way of example, autoimmune diseases such as idiopathicthrombocytopenia purpura (ITP), systemic lupus erythematosus (SLE), type1 diabetes mellitus, multiple sclerosis, aplastic anemia, psoriasis andrheumatoid arthritis.

Another significant therapeutic indication of the subject anti-B7.1antibodies is for prevention of graft-versus-host-disease (GVHD) duringorgan transplant and bone marrow transplant (BMT). The subjectantibodies may be used to induce host tolerance to donor-specificalloantigens and thereby facilitate engraftment and reduce the incidenceof graft rejection. It has been shown in a murine model of allogeneiccardiac transplantation that intravenous administration of CTLA4-Ig canresult in immunosuppression or even induction of tolerance toalloantigen. (Lin et al, J. Exp. Med. 178:1801, 1993; Torka et al, Proc.Natl. Acad. Sci., USA, 89:11102, 1992). It is expected that the subjectprimatized anti-B7.1 antibodies will exhibit similar or greateractivity.

Antibodies produced in the manner described above, or by equivalenttechniques, can be purified by a combination of affinity and sizeexclusion chromatography for characterization in functional biologicalassays. These assays include determination of specificity and bindingaffinity as well as effector function associated with the expressedisotype, e.g., ADCC, or complement fixation. Such antibodies may be usedas passive or active therapeutic agents against a number of humandiseases, including B cell lymphoma, infectious diseases including AIDS,autoimmune and inflammatory diseases, and transplantation. Theantibodies can be used either in their native form, or as part of anantibody/chelate, antibody/drug or antibody/toxin complex. Additionally,whole antibodies or antibody fragments (Fab₂, Fab, Fv) may be used asimaging reagents or as potential vaccines or immunogens in activeimmunotherapy for the generation of anti-idiotypic responses.

The amount of antibody useful to produce a therapeutic effect can bedetermined by standard techniques well known to those of ordinary skillin the art. The antibodies will generally be provided by standardtechnique within a pharmaceutically acceptable buffer, and may beadministered by any desired route. Because of the efficacy of thepresently claimed antibodies and their tolerance by humans it ispossible to administer these antibodies repetitively in order to combatvarious diseases or disease states within a human.

The anti-B7.1 antibodies (or fragments thereof) of this invention areuseful for inducing immunosuppression, i.e., inducing a suppression of ahuman's or animal's immune system. This invention therefore relates to amethod of prophylactically or therapeutically inducing immunosuppressionin a human or other animal in need thereof by administering aneffective, non-toxic amount of such an antibody of this invention tosuch human or other animal.

The ability of the compounds of this invention to induceimmunosuppression has been demonstrated in standard tests used for thispurpose, for example, a mixed lymphocyte reaction test or a testmeasuring inhibition of T-cell proliferation measured by thymidineuptake.

The fact that the antibodies of this invention have utility in inducingimmunosuppression indicates that they should be useful in the treatmentor prevention of resistance to or rejection of transplanted organs ortissues (e.g., kidney, heart, lung, bone marrow, skin, cornea, etc.);the treatment or prevention of autoimmune, inflammatory, proliferativeand hyperproliferative diseases, and of cutaneous manifestations ofimmunologically medicated diseases (e.g., rheumatoid arthritis, lupuserythematosus, systemic lupus erythematosus, Hashimotos thyroiditis,multiple sclerosis, myasthenia gravis, type 1 diabetes, uveitis,nephrotic syndrome, psoriasis, atopical dermatitis, contact dermatitisand further eczematous dermatitides, seborrheic dermatitis, Lichenplanus, Pemplugus, bullous pemphigus, Epidermolysis bullosa, urticaria,angioedemas, vasculitides, erythema, cutaneous eosinophilias, Alopeciaareata, etc.); the treatment of reversible obstructive airways disease,intestinal inflammations and allergies (e.g., Coeliac disease,proctitis, eosinophilia gastroenteritis, mastocytosis, Crohn's diseaseand ulcerative colitis) and food-related allergies (e.g., migraine,rhinitis and eczema).

One skilled in the art would be able, by routine experimentation, todetermine what an effective, non-toxic amount of antibody would be forthe purpose of inducing immunosuppression. Generally, however, aneffective dosage will be in the range of about 0.05 to 100 milligramsper kilogram body weight per day.

The antibodies (or fragments thereof) of this invention should also beuseful for treating tumors in a mammal. More specifically, they shouldbe useful for reducing tumor size, inhibiting tumor growth and/orprolonging the survival time of tumor-bearing animals. Accordingly, thisinvention also relates to a method of treating tumors in a human orother animal by administering to such human or animal an effective,non-toxic amount of an antibody. One skilled in the art would be able,by routine experimentation, to determine what an effective, non-toxicamount of anti-B7 antibody would be for the purpose of treatingcarcinogenic tumors. Generally, however, an effective dosage is expectedto be in the range of about 0.05 to 100 milligrams per kilogram bodyweight per day.

The antibodies of the invention may be administered to a human or otheranimal in accordance with the aforementioned methods of treatment in anamount sufficient to produce such effect to a therapeutic orprophylactic degree. Such antibodies of the invention can beadministered to such human or other animal in a conventional dosage formprepared by combining the antibody of the invention with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.

The route of administration of the antibody (or fragment thereof) of theinvention may be oral, parenteral, by inhalation or topical. The termparenteral as used herein includes intravenous, intraperitoneal,intramuscular, subcutaneous, rectal or vaginal administration. Thesubcutaneous and intramuscular forms of parenteral administration aregenerally preferred.

The daily parenteral and oral dosage regimens for employing compounds ofthe invention to prophylactically or therapeutically induceimmunosuppression, or to therapeutically treat carcinogenic tumors willgenerally be in the range of about 0.05 to 100, but preferably about 0.5to 10, milligrams per kilogram body weight per day.

The antibodies of the invention may also be administered by inhalation.By “inhalation” is meant intranasal and oral inhalation administration.Appropriate dosage forms for such administration, such as an aerosolformulation or a metered dose inhaler, may be prepared by conventionaltechniques. The preferred dosage amount of a compound of the inventionto be employed is generally within the range of about 10 to 100milligrams.

The antibodies of the invention may also be administered topically. Bytopical administration is meant non-systemic administration and includesthe application of an antibody (or fragment thereof) compound of theinvention externally to the epidermis, to the buccal cavity andinstillation of such an antibody into the ear, eye and nose, and whereit does not significantly enter the blood stream. By systemicadministration is meant oral, intravenous, intraperitoneal andintramuscular administration. The amount of an antibody required fortherapeutic or prophylactic effect will, of course, vary with theantibody chosen, the nature and severity of the condition being treatedand the animal undergoing treatment, and is ultimately at the discretionof the physician. A suitable topical dose of an antibody of theinvention will generally be within the range of about 1 to 100milligrams per kilogram body weight daily.

Formulations

While it is possible for an antibody or fragment thereof to beadministered alone, it is preferable to present it as a pharmaceuticalformulation. The active ingredient may comprise, for topicaladministration, from 0.001% to 10% w/w, e.g., from 1% to 2% by weight ofthe formulation, although it may comprise as much as 10% w/w butpreferably not in excess of 5% w/w and more preferably from 0.1% to 1%w/w of the formulation.

The topical formulations of the present invention, comprise an activeingredient together with one or more acceptable carrier(s) therefor andoptionally any other therapeutic ingredients(s). The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of where treatment is required, such as liniments, lotions,creams, ointments or pastes, and drops suitable for administration tothe eye, ear or nose.

Drops according to the present invention may comprise sterile aqueous oroily solutions or suspensions and may be prepared by dissolving theactive ingredient in a suitable aqueous solution of a bactericidaland/or fungicidal agent and/or any other suitable preservative, andpreferably including a surface active agent. The resulting solution maythen be clarified by filtration, transferred to a suitable containerwhich is then sealed and sterilized by autoclaving or maintaining at90°-100° C. for half an hour. Alternatively, the solution may besterilized by filtration and transferred to the container by an aseptictechnique. Examples of bactericidal and fungicidal agents suitable forinclusion in the drops are phenylmercuric nitrate or acetate (0.002%),benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%).Suitable solvents for the preparation of an oily solution includeglycerol, diluted alcohol and propylene glycol.

Lotions according to the present invention include those suitable forapplication to the skin or eye. An eye lotion may comprise a sterileaqueous solution optionally containing a bactericide and may be preparedby methods similar to those for the preparation of drops. Lotions orliniments for application to the skin may also include an agent tohasten drying and to cool the skin, such as an alcohol or acetone,and/or a moisturizer such as glycerol or an oil such as castor oil orarachis oil.

Creams, ointments or pastes according to the present invention aresemi-solid formulations of the active ingredient for externalapplication. They may be made by mixing the active ingredient infinely-divided or powdered form, alone or in solution or suspension inan aqueous or non-aqueous fluid, with the aid of suitable machinery,with a greasy or non-greasy basis. The basis may comprise hydrocarbonssuch as hard, soft or liquid paraffin, glycerol, beeswax, a metallicsoap; a mucilage; an oil of natural origin such as almond, corn,arachis, castor or olive oil; wool fat or its derivatives, or a fattyacid such as stearic or oleic acid together with an alcohol such aspropylene glycol or macrogols. The formulation may incorporate anysuitable surface active agent such as an anionic, cationic or non-ionicsurface active such as sorbitan esters or polyoxyethylene derivativesthereof. Suspending agents such as natural gums, cellulose derivativesor inorganic materials such as silicaceous silicas, and otheringredients such as lanolin, may also be included.

The subject anti-B7.1 antibodies or fragments thereof may also beadministered in combination with other moieties which modulate theB7:CD28 pathway. Such moieties include, by way of example, cytokinessuch as IL-7 and IL-10, CTLA4-Ig, soluble CTLA-4 and anti-CD28antibodies and fragments thereof.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of an antibody or fragmentthereof of the invention will be determined by the nature and extent ofthe condition being treated, the form, route and site of administration,and the particular animal being treated, and that such optimums can bedetermined by conventional techniques. It will also be appreciated byone of skill in the art that the optimal course of treatment, i.e., thenumber of doses of an antibody or fragment thereof of the inventiongiven per day for a defined number of days, can be ascertained by thoseskilled in the art using conventional course of treatment determinationtests.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following formulations are, therefore, to beconstrued as merely illustrative embodiments and not a limitation of thescope of the present invention in any way.

Capsule Composition

A pharmaceutical composition of this invention in the form of a capsuleis prepared by filling a standard two-piece hard gelatin capsule with 50mg. of an antibody or fragment thereof of the invention, in powderedform, 100 mg. of lactose, 32 mg. of talc and 8 mg. of magnesiumstearate.

Injectable Parenteral Composition

A pharmaceutical composition of this invention in a form suitable foradministration by injection is prepared by stirring 1.5% by weight of anantibody or fragment thereof of the invention in 10% by volume propyleneglycol and water. The solution is sterilized by filtration.

Ointment Composition

Antibody or fragment thereof of the invention 1.0 g.

White soft paraffin to 100.0 g.

The antibody or fragment thereof of the invention is dispersed in asmall volume of the vehicle to produce a smooth, homogeneous product.Collapsible metal tubes are then filled with the dispersion.

Topical Cream Composition

Antibody or fragment thereof of the invention 1.0 g.

Polawax GP 200 20.0 g.

Lanolin Anhydrous 2.0 g.

White Beeswax 2.5 g.

Methyl hydroxybenzoate 0.1 g.

Distilled Water to 100.0 g.

The polawax, beeswax and lanolin are heated together at 60° C. Asolution of methyl hydroxybenzoate is added and homogenization isachieved using high speed stirring. The temperature is then allowed tofall to 50° C. The antibody or fragment thereof of the invention is thenadded and dispersed throughout, and the composition is allowed to coolwith slow speed stirring.

Topical Lotion Composition

Antibody or fragment thereof of the invention 1.0 g.

Sorbitan Monolaurate 0.6 g.

Polysorbate 20 0.6 g.

Cetostearyl Alcohol 1.2 g.

Glycerin 6.0 g.

Methyl Hydroxybenzoate 0.2 g.

Purified Water B.P. to 100-00 ml. (B.P.=British Pharmacopeia)

The methyl hydroxybenzoate and glycerin are dissolved in 70 ml. of thewater at 75° C. The sorbitan monolaurate, polysorbate 20 and cetostearylalcohol are melted together at 75° C. and added to the aqueous solution.The resulting emulsion is homogenized, allowed to cool with continuousstirring and the antibody or fragment thereof of the invention is addedas a suspension in the remaining water. The whole suspension is stirreduntil homogenized.

Eye Drop Composition

Antibody or fragment thereof of the invention 0.5 g.

Methyl Hydroxybenzoate 0.01 g.

Propyl Hydroxybenzoate 0.04 g.

Purified Water B.P. to 100-00 ml.

The methyl and propyl hydroxybenzoates are dissolved in 70 ml. purifiedwater at 75° C. and the resulting solution is allowed to cool. Theantibody or fragment thereof of the invention is then added, and thesolution is sterilized by filtration through a membrane filter (0.022 μmpore size), and packed aseptically into suitable sterile containers.

Composition for Administration by Inhalation

For an aerosol container with a capacity of 15-20 ml: mix 10 mg. of anantibody or fragment thereof of the invention with 0.2-0.5% of alubricating agent, such as polysorbate 85 or oleic acid, and dispersesuch mixture in a propellant, such as freon, preferably in a combinationof (1,2 dichlorotetrafluoroethane) and difluorochloro-methane and putinto an appropriate aerosol container adapted for either intranasal ororal inhalation administration.

Composition for Administration by Inhalation

For an aerosol container with a capacity of 15-20 ml: dissolve 10 mg. ofan antibody or fragment thereof of the invention in ethanol (6-8 ml.),add 0.1-0.2% of a lubricating agent, such as polysorbate 85 or oleicacid; and disperse such in a propellant, such as freon, preferably incombination of (1.2 dichlorotetrafluoroethane) anddifluorochloromethane, and put into an appropriate aerosol containeradapted for either intranasal or oral inhalation administration.

The antibodies and pharmaceutical compositions of the invention areparticularly useful for parenteral administration, i.e., subcutaneously,intramuscularly or intravenously. The compositions for parenteraladministration will commonly comprise a solution of an antibody orfragment thereof of the invention or a cocktail thereof dissolved in anacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers may be employed, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like. These solutions are sterile and generally free ofparticulate matter. These solutions may be sterilized by conventional,well-known sterilization techniques. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as Ph adjusting and bufferingagents, etc. The concentration of the antibody or fragment thereof ofthe invention in such pharmaceutical formulation can vary widely, i.e.,from less than about 0.5%, usually at or at least about 1% to as much asis i5 or 20% by weight, and will be selected primarily based on fluidvolumes, viscosities, etc., according to the particular mode ofadministration selected.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 Ml sterile buffered water, and50 mg. of an antibody or fragment thereof of the invention. Similarly, apharmaceutical composition of the invention for intravenous infusioncould be made up to contain 250 ml. of sterile Ringer's solution, and150 mg. of an antibody or fragment thereof of the invention. Actualmethods for preparing parenterally administrable compositions are wellknown or will be apparent to those skilled in the art, and are describedin more detail in, for example, Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa., hereby incorporated byreference herein.

The antibodies (or fragments thereof) of the invention can belyophilized for storage and reconstituted in a suitable carrier prior touse. This technique has been shown to be effective with conventionalimmune globulins and art-known lyophilization and reconstitutiontechniques can be employed.

Depending on the intended result, the pharmaceutical composition of theinvention can be administered for prophylactic and/or therapeutictreatments. In therapeutic application, compositions are administered toa patient already suffering from a disease, in an amount sufficient tocure or at least partially arrest the disease and its complications. Inprophylactic applications, compositions containing the presentantibodies or a cocktail thereof are administered to a patient notalready in a disease state to enhance the patient's resistance.

Single or multiple administrations of the pharmaceutical compositionscan be carried out with dose levels and pattern being selected by thetreating physician. In any event, the pharmaceutical composition of theinvention should provide a quantity of the altered antibodies (orfragments thereof) of the invention sufficient to effectively treat thepatient.

It should also be noted that the antibodies of this invention may beused for the design and synthesis of either peptide or non-peptidecompounds (mimetics) which would be useful in the same therapy as theantibody. See, e.g., Saragovi et al., Science, 253, 792-795 (1991).

To further illustrate the invention, the following examples areprovided. These examples are not intended, nor are they to be construed,as further limiting the invention.

EXAMPLE 1

Recombinant immunoglobulin libraries displayed on the surface offilamentous phage were first described by McCafferty et al, Nature,348:552-554, 1990 and Barbas et al, Proc. Natl. Acad. Sci., USA88:7978-7982, 1991. Using this technology, high affinity antibodies havebeen isolated from immune human recombinant libraries (Barbas et al,Proc. Natl. Acad. Sci., USA 589:10164-10168, 1992). Although the phagedisplay concept used is substantially similar to that described byBarbas, 1991, Id. the technique has been modified by the substitution ofa unique vector for monkey libraries to reduce the possibility ofrecombination and improve stability. This vector, pMS, FIG. 1 contains asingle lac promoter/operator for efficient transcription and translationof polycistronic heavy and light chain monkey DNA. This vector containstwo different leader sequences, the omp A (Movva et al, J. Biol. Chem.,255: 27-29, (1980), for the light chain and the pel B (Lei, J. Bact.,4379-109:4383 (1987) for the heavy chain Fd. Both leader sequences aretranslated into hydrophobic signal peptides that direct the secretion ofthe heavy and light chain cloned products into the periplasmic space. Inthe oxidative environment of the periplasm, the two chains fold anddisulfide bonds form to create stable Fab fragments. We derived thebackbone of the vector from the phagemid bluescript. (Stratagene, LaJolla, Calif.). It contains the gene for the enzyme beta-lactamase thatconfers ampicillin (carbenicillin) resistance to bacteria that harborpMS DNA. We also derived, from bluescript, the origin of replication ofthe multicopy plasmid ColEl and the origin of replication of thefilamentous bacteriophage f1. The origin of replication of phage f1 (theso-called intragenic region), signals the initiation of synthesis ofsingle stranded pMS DNA, the initiation of capsid formation and thetermination of RNA synthesis by viral enzymes. The replication andassembly of pMS DNA strands into phage particles requires viral proteinsthat must be provided by a helper phage. We have used helper phageVCSM13 which is particularly suited for this, since it also contains agene coding for kanamycin resistance. Bacteria infected with VCSM13 andpMS can be selected by adding both kanamycin and carbenicillin to thegrowth medium. The bacteria will ultimately produce filamentous phageparticles containing either pMS or VCSM13 genomes. Packaging of thehelper phage is less efficient than that of pMS, resulting in a mixedphage population that contains predominately recombinant pMS phages. Theends of the phage pick up minor coat proteins specific to each end. Ofparticular interest here is the gene III product which is present inthree to five copies at one end of the phage. The gene III product is406 amino acid residues and is required for phage infection of E. colivia the F pili. The first two domains of the heavy chain, the variableand the CH1 domain, are fused to the carboxy-terminal half of the geneIII protein. This recombinant pili protein, directed by the pel Bleader, is secreted to the peroplasm where it accumulates and formsdisulfide bonds with the light chain before it is incorporated in thecoat of the phage. Also, another vector contains a FLAG sequenceengineered downstream of the gene III. The FLAG is an 8 amino acidpeptide expressed at the carboxy terminal of the Fd protein. We areusing commercially available monoclonal anti-FLAG M2 for bothpurification and detection of phage Fab by ELISA (Brizzard, BioTechniques, 16(4):730-731, (1994)).

After constructing the vector pMS, we tested its ability to producephage bound Fab using control antibody genes. We cloned an anti-tetanustoxoid antibody, (obtained from Dr. Carlos Barbas), into pMS andtransformed XLI-blue. We co-infected our cells with VCSM13 and generatedphage displaying the anti-tetanus toxoid antibody. We performedefficiency experiments where anti-tetanus toxoid phage were combinedwith phage beading an irrelevant antibody at 1:100,000. We performedthree rounds of panning by applying 50 μl of the mixed phage to antigen(tetanus toxoid) coated polystyrene wells. Non-adherent phage werewashed off and the adherent phage were eluted with acid. The elutedphage were used to infect a fresh aliquot of XL1-Blue bacteria andhelper phage was added. After overnight amplification, phage wereprepared and again panned on antigen coated plates. After three roundsof panning, we were able to show that we had successfully enriched forthe anti-tetanus toxoid phage. The success of this technology alsodepends on the ability to prepare soluble Fabs for characterization ofthe final panned product. This was achieved by excising gene III fromthe pMS DNA using the restriction enzyme Nhe I followed by re-ligation.After the gene III was excised, the Fab was no longer displayed on thephage surface but accumulated in the piroplasmic space. Lysates wereprepared from bacteria expressing soluble Fab and tested for antigenspecificity using an ELISA. High levels of soluble Fab were detected.

In order to adapt phage display technology for use with macaquelibraries, we developed specific primers for PCR amplifying monkeyimmunoglobulin genes. These were based on macaque sequences we obtainedwhile developing the PRIMATIZED™ antibody technology (See, Ser. No.08/379,072, incorporated by reference herein) and databases containinghuman sequences. (Kabat et al, (1991), “Sequences of Proteins ofImmunological Interest,” U.S. Dept. of Health and Human Services,National Institute of Health).

We developed three sets of primers to cover amplification of the macaquerepertoire. Our first set of primers was designed for amplification ofthe heavy chain VH and CH1 (Fd) domains. It consisted of a 3′ CH1 domainprimer and six 5′ VH family specific primers that bind in the framework1 region. Our second set of primers, for amplifying the whole lambdachain, covers the many lambda chain subgroups. It consists of a 3′primer and three 5′ degenerate primers that bind in the VL framework 1region. Our third set of primers was designed for amplification of thekappa chain subgroups. It consists of one 3′ primer and five VKframework 1 primers. Using each of these sets, PCR parameters wereoptimized to obtain strong enough signals from each primer pair so thatample material was available for cloning of the library. We recentlycreated macaque combinatorial libraries in our pMS vector using theseoptimized PCR conditions. Bone marrow biopsies were taken from CD4immune monkeys as the source of immunoglobulin RNA. The librariescontained approximately 10⁶ members and are currently being panned forspecific binders on antigen coated wells.

EXAMPLE 2

Development of B7/CTLA-4 Reagents

We have generated a number of reagents for the purpose of immunizingmonkeys, developing binding and functional assays in vitro, screeningheterohybridomas and panning phage libraries. Table 1 lists each reagentand its intended purpose. In the case of B7.1, RNA was extracted from SBcells and converted to cDNA using reverse transcriptase. The firststrand cDNA was PCR amplified using B7.1 specific primers and clonedinto IDEC's NEOSPLA mammalian expression vectors. CHO cells weretransfected with B7.1 NEOSPLA DNA and clones expressing membraneassociated B7.1 were identified. The B7.1 fusion protein was generatedsimilarly, except that the PCR amplified B7.1 gene was cloned into aNEOSPLA cassette vector containing the human CH2 and CH3 immunoglobulingenes. CHO cells were transformed with the B7.1/Ig NEOSPLA DNA andstable clones secreting B7.1/Ig fusion protein were amplified. Ingeneral, the B7.2 and CTLA4 reagents were generated in the same manner,except that for B7.2 the RNA was isolated from human spleen cells thathad been stimulated 24 hours with anti-Ig and IL-4, and for the CTLA4constructs the gene source was PHA activated human T cells.

TABLE 1 Reagent Purpose CHO Expression Soluble B7.1 Immunization,immunoassays Yes B7.1 Transfectant Screening, ELISA Yes B7.1/Ig FusionProtein Inhibition studies, panning Yes B7.2 Transfectant Screening,ELISA Yes B7.2/Ig Fusion Protein Inhibition studies, panning To becompleted CTLA4 Transfectant Inhibition studies To be completed CTLA4/IgInhibition studies To be completed

The availability of these reagents, together with monoclonal antibodiesto B7.1 (L3074) (Becton Dickinson, 1994) and B7.2 (Fun-1 (Engel et al,Blood, 84, 1402-1407, (1994) and purified goat and rabbit antisera,specifically developed to detect monkey Fab fragments, facilitatesidentification of antibodies having the desired properties.

EXAMPLE 3

Investigation of the Immune Response in Cynomolgus Monkeys to Solubleand Cell Associated Human B7.1

To evaluate the feasibility of producing monkey antibodies to human B7.1antigen, we first purified recombinant SB7.1 from CHO cell media byaffinity chromatography using a L307.4-sepharose affinity column. SB7.1was then injected, with adjuvant, into five mature cynomolgus macaques.After a 3 to 4 month period of booster immunizations, sera from themonkeys immunized with SB7.1 or human SE cells were tested for antigenbinding.

Serum samples from the five monkeys immunized with SB7.1. and threeadditional animals immunized with B7.1 positive human SB cells, weretested for antibody titers against membrane associated B7.1 expressed intransfected CHO cells. The results summarized in FIG. 3 showed that fourout of five monkeys immunized with affinity-purified SB7.1 producedantibody titers in excess of 1:5000. The three animals immunized with SBcells containing cell associated B7.1 expressed lower titers ofantibodies ranging from 1:1400 to 1:2800.

EXAMPLE 4

We purified antibodies from sera of all eight immunized monkeys usingSB7.1-sepharose and then tested their ability to bind to 1) SB7.1 coatedplates in ELISA; 2) antigen positive B cells and 3) B7.1 CHOtransfectomas. In addition, they were evaluated for their ability toblock B cell interactions as measured by IL-2 production and tritiatedthymidine uptake in a mixed lymphocyte reaction (MLR). For T cellbinding experiments, human buffy coat peripheral blood lymphocytes werecultured for 3-6 days in the presence of PHA stimulator. B7 binding wasdetected by radio assay using ¹²⁵I-radiolabeled soluble B7.1 (SB7.1).

EXAMPLE 5

Direct Binding of Monkey Antibodies to Radiolabeled SB7 ¹²⁵Iradiolabeled SB7.1 was tested for binding to anti-B7.1 antibodies at 4,1 and 0.25 μg/ml in solution. The results shown in Table 2 suggest thatmost of the antibodies produced by monkeys immunized with SB7.1 werecapable of binding the affinity-purified ¹²⁵I-SB7.1 in a concentrationdependent manner. To evaluate the specificity of binding to labeledSB7.1, unlabelled concentration dependent manner. To evaluate thespecificity of binding to labeled SB7.1, unlabelled SB7.1 competitionexperiments were done with antibodies from two animals.Affinity-purified antibodies from monkeys 1133 and 1144 were coated ontomicrowell plates at 400 ng/well. Affinity-purified unlabeled SB7.1 (500and 100 ng/well) was used as competitor. The results shown in FIG. 4demonstrated that SB7.1 preparations are effective in inhibiting the¹²⁵I-SB7.1 from binding to the antibodies.

TABLE 2 Binding of SB7-I¹²⁵ to Monkey Antibodies Affinity Purified on aSB7-Sepharose Affinity Column Antibody Monkey Numbers (μg/ml) 769 9081133 1135 1137 1139 1144 1146 4 175 213 9,056 12,771 4,318 226 5,781108  1 106 142 6,569  7,940 3,401 110 3,901 80 0.25  95 104 1,803  2,6731,219 100 1,186 94 Data are mean values of duplicate assays andrepresent cpm SB7-I¹²⁵ bound.

EXAMPLE 6

Direct Binding of Radiolabeled Affinity-purified Monkey Antibodies toB7⁺ Cells and Inhibition by SB7.1

Affinity-purified radiolabeled monkey anti-B7.1 antibodies from monkeyPRI135 were compared with radiolabeled L307.4 MAb for direct binding toB7 positive human SB cells. As a specificity control, unlabeled SB7.1(0.002-20 μg/mi) was added to compete with both radiolabeled antibodies.We demonstrated that monkey antibodies can bind cell associated B7.1 andare inhibited with SB7.1, as shown in FIG. 5. Inhibition as high as 90%was observed with SB7.1.

EXAMPLE 7

Direct Binding of Radiolabeled B7-Ig Fusion Protein to Activated T Cellsand Inhibition by Affinity-purified Monkey Antibodies

Human peripheral blood T lymphocytes were activated for 3-6 days andtested for direct binding of ¹²⁵I-B7.1-Ig. Because of Fc receptorupregulation on activated human T cells, it was necessary topre-incubate the cells with heat-aggregated pre-immune immunoglobulin toblock Fc binding sites prior to addition of B7.1-Ig to the cells. Abackground control using SP2/0 murine myeloma cells was included toallow correction of the background binding. FIG. 6 shows that inhibitionof ¹²⁵I-B7.1-Ig fusion protein binding to activated T cells was achievedwith affinity-purified monkey antibodies at concentrations from 200 to 8μg/ml. Unlabeled SB7.1 and L307.4 MAb used as controls were alsoeffective in inhibiting B7.1-Ig fusion protein cell binding.

EXAMPLE 8

Inhibition of IL-2 Production in Mixed Lymphocyte Reactions by MonkeyAnti-B7 Antibodies

The blocking of CD28/B7 interaction leads to inhibition of IL-2production by T lymphocytes. In the experiment shown in FIG. 7,affinity-purified monkey antibodies from two monkeys immunized withSB7.1 (monkeys 1137 and 1135) and one immunized with B7 positive SBcells (monkey 1146) were evaluated for their abilities to inhibit humanT cell activation in mixed lymphocyte reaction (MLR), as measured byinhibition of IL-2 production. The results of this experiment show thataffinity-purified anti-B7.1 antibodies from monkeys 1146 and 1137inhibited IL-2 production when added at concentrations of 50 μg/ml.Monkey 1135 antibodies could not be evaluated at the two highestconcentrations due to lack of material, yet gave significant inhibitionat lower concentrations. The murine MAb L307.4 was inhibitory atconcentrations of 10 μg/ml. Other monkey sera tested at theseconcentrations were negative (data not shown). These results demonstratethat at least three of the monkeys immunized with both soluble andmembrane associated forms of the B7 antigen are producing B7-blockingantibodies with immunosuppressive potential.

EXAMPLE 9

Investigation of Cross-reactivity in B7.1 Immunized Monkey Serum to B7.2Antigen

Antibodies raised against B7.1 are to be tested for cross-reactivity toB7.2. Preliminary results using B7.1 affinity-purified antibodies fromB7.1 immune sera provided suggestive evidence of binding to B7.2transfected CHO cells (not shown). These data should be confirmed byusing soluble B7.2Ig reagents. We will first purify additional monkeyantibodies from B7.1 immunized animals by affinity chromatography onB7.1Ig-sepharose. We will then produce and purify B7.2Ig from CHO cellsin sufficient quantities to prepare a B7.2Ig-sepharose affinity column.We will select from the B7.1 specific antibody population thoseantibodies which cross-react with B7.2 by binding to theB7.2Ig-sepharose column. Any cross-reactive antibodies identified willbe further characterized by direct binding to both B7.1 and B7.2transfected CHO cells and inhibition of binding to B7.2 transfectedcells by B7.1Ig.

EXAMPLE 10

Generation of a Phage Display Library

Recombinant phage display libraries are generated from B7.1 and B7.2immune monkeys. Lymph node and bone marrow biopsies are performed 7-12days after immunization to harvest RNA rich B cells and plasma cells.RNA is isolated from the lymphocytes using the method described byChomczynski Anal. Biochem., 162(1), 156-159, (1987). RNA is converted tocDNA using an oligo dT primer and reverse transcriptase. The firststrand cDNA is divided into aliquots and PCR amplified using the sets ofkappa, lambda, and heavy chain Fd region primers described earlier andeither Pfu polymerase (Stratagene, San Diego) or Taq polymerase(Promega, Madison). The heavy chain PCR amplified products are pooled,cut with Xho VSpe I restriction enzymes and cloned into the vector pMS.Subsequently, the light chain PCR products are pooled, cut with SacI/Xba I restriction enzymes, and cloned to create the recombinantlibrary. XLI-Blue E. coli is transformed with the library DNA andsuper-infected with VCSM13 to produce the phage displaying antibodies.The library is panned four rounds on polystyrene wells coated with B7.1or B7.2 antigen. Individual phage clones from each round of panning areanalyzed. The pMS vector DNA is isolated and the gene III excised.Soluble Fab fragments are generated and tested in ELISA for binding toB7.1 and B7.2.

EXAMPLE 11

Characterization of Phage Fab Fragments

The monkey phage Fab fragments are characterized for their specificityand the ability to block B7.1-Ig and B7.2-Ig binding to CTLA-4-Ig orCTLA-4 transfected cells. Phage fragments are also characterized forcross-reactivity after first panning for 4 rounds on the B7 species usedfor immunization in order to select for high affinity fragments. Fabfragments identified from four rounds of panning either on B7.1 or B7.2antigen coated surfaces are scaled up by infection and grown in 24 hourfermentation cultures of E coli. Fragments are purified by Kodak FLAGbinding to a anti-FLAG affinity column. Purified phage Fabs are testedfor affinity by an ELISA based direct binding modified Scatchardanalysis (Katoh et al, J. Chem. BioEng., 76:451-454, (1993)) using Goatanti-monkey Fab antibodies or anti-FLAG MAb conjugated with horseradishperoxidase. The anti-monkey Fab reagents will be absorbed against humanheavy chain constant region Ig to remove any cross-reactivity to B7-Ig.Kd values are calculated for each fragment after measurements of directbinding to B7.1-Ig or B7.2-Ig coated plates.

EXAMPLE 12

Phage Fab Fragment Blocking of CTLA-4/B7 Binding

Fab fragments most effectively blocking the binding of B7-Ig at thelowest concentrations are selected as lead candidates. Selections aremade by competing off ¹²⁵I-B7-Ig binding to CTLA-4-Ig or CTLA-4transfected cells. Additional selection criteria include, blocking ofmixed lymphocyte reaction (MLR), as measured by inhibiting 3H-thymidineuptake in responder cells (Azuma et al, J. Exp. Med., 177:845-850,;Azuma et al, Nature, 301:76-79, (1993)) and direct analysis of IL-2production using IL-2 assay kits. The three or four candidates which aremost effective in inhibiting of MLR and CTLA-4 binding assays are chosenfor cloning into the above-described mammalian expression vector fortransfection into CHO cells and expression of chimeric monkey/humanantibodies.

EXAMPLE 13

Generation of Monkey Heterohybridomas

Monkey heterohybridomas secreting monoclonal antibodies are generatedfrom existing immunized animals whose sera tested positive for B7.1and/or B7.2. Lymph node biopsies are taken from animals positive toeither, or both, antigens. The method of hybridoma production is similarto the established method used for the generation of monkey anti-CD4antibodies (Newman, 1992(Id.)). Monkeys with high serum titers will havesections of inguinal lymph nodes removed under anesthesia. Lymphocytesare washed from the tissue and fused with KH6/B5 heteromyeloma cells(Carrol et al, J. Immunol. Meth., 89:61-72, (1986)) using polyethyleneglycol (PEG). Hybridomas are selected on H.A.T. media and stabilized byrepeated subcloning in 96 well plates.

Monkey monoclonal antibodies specific for B7.1 antigen are screened forcross-reactivity to B7.2. Monkey anti-B7 antibodies will becharacterized for blocking of B7/CTLA-4 binding using the ¹²⁵I-B7-Igbinding assay. Inhibition of MLR by 3H-Thymidine uptake and directmeasurement of IL-2 production is used to select three candidates. Twocandidates will be brought forward in Phase II studies and expressed inCHO cells while repeating all functional studies. For the purposes ofdeveloping an animal model for in vivo pharmacology, anti-B7 antibodieswill be tested on cells of several animal species. The establishment ofan animal model will allow preclinical studies to be carried out for theselected clinical indication.

EXAMPLE 14

As discussed supra, using the above heterohybridoma methods, 4 leadmonkey anti-B7.1 antibodies have been identified: 16C10, 7B6, 7C10 and20C9. These antibodies were characterized as follows:

To demonstrate the monkey antibodies' ability to block the physicalinteraction between CTLA4-Ig, varying concentrations of the monkeyanti-B7.1 antibodies and unlabeled CTLA4-Ig were incubated withradiolabeled CTLA4-Ig^(I125). The results of the inhibition assay showedthat the IC50 (the concentration of inhibitor which results in 50%inhibition) for the monkey antibodies are:

a: 7C10: 0.39 μg/Ml b: 16C10: 1.60 μg/Ml c: 20C9: 3.90 μg/Ml d: 7B6:39.0 μg/Ml

Scatchard analysis showed that the apparent affinity constants (Kd) forthe monkey antibodies binding to B7-Ig coated plates were approximatedto be:

a: 7C10:  6.2 × 10⁻⁹M b: 16C10:  8.1 × 10⁻⁹M c: 7B6: 10.7 × 10⁻⁹M d:20C9: 16.8 × 10⁻⁹M

The antibodies were tested in vitro in a mixed lymphocyte reaction assay(MLR). The MLR showed that all 4 anti-B7.1 antibodies inhibit IL-2production to different extents:

a: 7B6: 5.0 μg/Ml b: 16C10: 0.1 μg/Ml c: 20C9: 2.0 μg/Ml d: 7C10: 5.0μg/Ml

The monkey anti-B7.1 antibodies were tested for their ability to bind B7on human peripheral blood lymphocytes (PBL). FACS analysis showed thatall 4 monkey antibodies tested positive.

Monkey antibodies 16C10, 7B6, 7C10 and 20C9 were tested for C1q bindingby FACS analysis. Results showed 7C10 monkey Ig had strong human C1qbinding after incubating with B7.1 CHO-transfected cells. 16C10 wasnegative, as were the 20C9 and 7B6 monkey antibodies.

EXAMPLE 15

Using the primatized antibody methodology incorporated by reference tocommonly assigned U.S. Ser. No. 08/379,072, and using the NEOSPLA vectorsystem shown in FIG. 2, the heavy and light variable domains of 7C10,7B6 and 16C10 were cloned and primatized forms thereof have beensynthesized in CHO cells using the NEOSPLA vector system. The amino acidand nucleic acid sequences for the primatized 7C10 light and heavychain, 7B6 light and heavy chain, and 16C10 light and heavy chain arerespectively shown in FIGS. 8 a, 8 b, 9 a, 9 b, 10 a and 10 b.

It is expected that these primatized antibodies, given their probablelow antigenicity and human effector function, will be well suited astherapeutics. In fact, it has recently been shown that primatized 16C10exhibits human C1₉ binding, whereas 16C10 does not.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be embraced by the following claims.

1. A method of treating B cell lymphoma comprising administering atherapeutically effective amount of an anti-CD80 antibody, wherein saidantibody binds the same epitope as an anti-CD80 antibody selected fromthe group consisting of 7C10, which has the variable region sequencesshown in FIGS. 8 a and 8 b (amino acid sequences of SEQ ID NOs. 1-2 and3-4 respectively); 7B6, which has the variable region sequences shown inFIGS. 9 a and 9 b (amino acid sequences of SEQ ID NOs. 5-6 and 7-3respectively); and 16C10, which has the variable region sequences shownin FIGS. 10 a and 10 b (amino acid sequences of SEQ ID NOs. 9-10 and11-12 respectively).
 2. The method of claim 1 wherein said anti-CD80antibody binds the same epitope as an anti-CD80 antibody selected fromthe group consisting of 16C10, 7C10, 20C9 and 7B6.
 3. A method oftreating B cell lymphoma comprising administering a therapeuticallyeffective amount of an anti-CD80 antibody, wherein said antibody bindsthe same epitope as an anti-CD80 antibody selected from the groupconsisting of 7C10, which has the variable region sequences shown inFIGS. 8 a and 8 b (amino acid sequences of SEQ ID NOs. 1-2 and 3-4respectively); 7B6, which has the variable region sequences shown inFIGS. 9 a and 9 b (amino acid sequences of SEQ ID NOs. 5-6 and 7-3respectively); and 16C10, which has the variable region sequences shownin FIGS. 10 a and 10 b (amino acid sequences of SEQ ID NOs. 9-10 and11-12 respectively), wherein said antibody is a non-depleting antibody.4. A method of treating B cell lymphoma comprising administering atherapeutically effective amount of an anti-CD80 antibody, wherein saidantibody is a primatized ® antibody having the variable region sequencesof an anti-CD80 antibody selected from the group consisting of 7C10,which has the variable region sequences shown in in FIGS. 8 a and 8 b(amino acid sequences of SEQ ID NOs. 1-2 and 3-4 respectively); 7B6,which has the variable region sequences shown in FIGS. 9 a and 9 b(amino acid sequences of SEQ ID NOs. 5-6 and 7-8, respectively); and16C10, which has the variable region sequences shown in FIGS. 10 a and10 b (amino acid sequences of SEQ ID NOs. 9-10 and 11-12 respectively).5. The method of claim 4 where the antibody is a primatized® antibodyhaving the variable region sequences of antibody 16C10 shown in FIGS. 10a and 10 b (amino acid sequences of SEQ ID NOs. 9-10 and 11-12,respectively).